Activation of Innate and Adaptive Immunity by a Recombinant Human Cytomegalovirus Strain Expressing an NKG2D Ligand.

Development of an effective vaccine against human cytomegalovirus (HCMV) is a need of utmost medical importance. Generally, it is believed that a live attenuated vaccine would best provide protective immunity against this tenacious pathogen. Here, we propose a strategy for an HCMV vaccine that aims at the simultaneous activation of innate and adaptive immune responses. An HCMV strain expressing the host ligand ULBP2 for the NKG2D receptor was found to be susceptible to control by natural killer (NK) cells, and preserved the ability to stimulate HCMV-specific T cells. Infection with the ULBP2-expressing HCMV strain caused diminished cell surface levels of MHC class I molecules. While expression of the NKG2D ligand increased the cytolytic activity of NK cells, NKG2D engagement in CD8+ T cells provided co-stimulation and compensated for lower MHC class I expression. Altogether, our data indicate that triggering of both arms of the immune system is a promising approach applicable to the generation of a live attenuated HCMV vaccine

Superior induction and maintenance of protective CD8 T cells in mice infected with mouse cytomegalovirus vector expressing RAE-1

Due to a unique pattern of CD8 T-cell response induced by cytomegaloviruses (CMVs), live attenuated CMVs are attractive candidates for vaccine vectors for a number of clinically relevant infections and tumors. NKG2D is one of the most important activating NK cell receptors that plays a role in costimulation of CD8 T cells. Here we demonstrate that the expression of CD8 T-cell epitope of Listeria monocytogenes by a recombinant mouse CMV (MCMV) expressing the NKG2D ligand retinoic acid early-inducible protein 1-gamma (RAE-1γ) dramatically enhanced the effectiveness and longevity of epitope-specific CD8 T-cell response and conferred protection against a subsequent challenge infection with Listeria monocytogenes. Unexpectedly, theattenuatedgrowth in vivo of the CMV vector expressing RAE-1γ and its capacity to enhance specific CD8 T-cell response were preserved even in mice lacking NKG2D, implying additional immune function for RAE-1γ beyond engagement of NKG2D. Thus, vectors expressing RAE-1γ represent a promising approach in the development of CD8 T-cell– based vaccine

Activation of innate and adaptive immunity by a recombinant human cytomegalovirus strain expressing an NKG2D ligand

Development of an effective vaccine against human cytomegalovirus (HCMV) is a need of utmost medical importance. Generally, it is believed that a live attenuated vaccine would best provide protective immunity against this tenacious pathogen. Here, we propose a strategy for an HCMV vaccine that aims at the simultaneous activation of innate and adaptive immune responses. An HCMV strain expressing the host ligand ULBP2 for the NKG2D receptor was found to be susceptible to control by natural killer (NK) cells, and preserved the ability to stimulate HCMV-specific T cells. Infection with the ULBP2-expressing HCMV strain caused diminished cell surface levels of MHC class I molecules. While expression of the NKG2D ligand increased the cytolytic activity of NK cells, NKG2D engagement in CD8+ T cells provided co-stimulation and compensated for lower MHC class I expression. Altogether, our data indicate that triggering of both arms of the immune system is a promising approach applicable to the generation of a live attenuated HCMV vaccine

IL-33/ST2 pathway drives regulatory T cell dependent suppression of liver damage upon cytomegalovirus infection

© 2017 Popovic et al. Regulatory T (Treg) cells dampen an exaggerated immune response to viral infections in order to avoid immunopathology. Cytomegaloviruses (CMVs) are herpesviruses usually causing asymptomatic infection in immunocompetent hosts and induce strong cellular immunity which provides protection against CMV disease. It remains unclear how these persistent viruses manage to avoid induction of immunopathology not only during the acute infection but also during life-long persistence and virus reactivation. This may be due to numerous viral immunoevasion strategies used to specifically modulate immune responses but also induction of Treg cells by CMV infection. Here we demonstrate that liver Treg cells are strongly induced in mice infected with murine CMV (MCMV). The depletion of Treg cells results in severe hepatitis and liver damage without alterations in the virus load. Moreover, liver Treg cells show a high expression of ST2, a cellular receptor for tissue alarmin IL-33, which is strongly upregulated in the liver of infected mice. We demonstrated that IL-33 signaling is crucial for Treg cell accumulation after MCMV infection and ST2-deficient mice show a more pronounced liver pathology and higher mortality compared to infected control mice. These results illustrate the importance of IL-33 in the suppressive function of liver Treg cells during CMV infection

NK/ILC1 cells mediate neuroinflammation and brain pathology following congenital CMV infection

Congenital human cytomegalovirus (cHCMV) infection of the brain is associated with a wide range of neurocognitive sequelae. Using infection of newborn mice with mouse cytomegalovirus (MCMV) as a reliable model that recapitulates many aspects of cHCMV infection, including disseminated infection, CNS infection, altered neurodevelopment, and sensorineural hearing loss, we have previously shown that mitigation of inflammation prevented alterations in cerebellar development, suggesting that host inflammatory factors are key drivers of neurodevelopmental defects. Here, we show that MCMV infection causes a dramatic increase in the expression of the microglia-derived chemokines CXCL9/CXCL10, which recruit NK and ILC1 cells into the brain in a CXCR3-dependent manner. Surprisingly, brain-infiltrating innate immune cells not only were unable to control virus infection in the brain but also orchestrated pathological inflammatory responses, which lead to delays in cerebellar morphogenesis. Our results identify NK and ILC1 cells as the major mediators of immunopathology in response to virus infection in the developing CNS, which can be prevented by anti-IFN-γ antibodies

Viral infection of the ovaries compromises pregnancy and reveals innate immune mechanisms protecting fertility

Viral infections during pregnancy are a considerable cause of adverse outcomes and birth defects, and the underlying mechanisms are poorly understood. Among those, cytomegalovirus (CMV) infection stands out as the most common intrauterine infection in humans, putatively causing early pregnancy loss. We employed murine CMV as a model to study the consequences of viral infection on pregnancy outcome and fertility maintenance. Even though pregnant mice successfully controlled CMV infection, we observed highly selective, strong infection of corpus luteum (CL) cells in their ovaries. High infection densities indicated complete failure of immune control in CL cells, resulting in progesterone insufficiency and pregnancy loss. An abundance of gap junctions, absence of vasculature, strong type I interferon (IFN) responses, and interaction of innate immune cells fully protected the ovarian follicles from viral infection. Our work provides fundamental insights into the effect of CMV infection on pregnancy loss and mechanisms protecting fertility

Encapsulated mesenchymal stem cells for in vivo immunomodulation

Mesenchymal stem cells (MSCs) are self-renewable, multipotent progenitor cells able to differentiate into various mesodermal lineages.1 MSCs are present in basically all tissues, and have a pivotal role in tissue repair and in local control of inflammation

Spread of the ULBP2-expressing HCMV strain is strongly controlled by NK cells.

<p>(A) The ULBP2-TB40 strain was generated by insertion of a ULBP2 expression cassette (driven by the mouse CMV major immediate-early promoter (mMIEP)), replacing ORF UL16 in the BAC-cloned TB40 genome that lacks also the US1 to US6 region. (B) HFF were infected at an MOI of 1 with TB40 or ULBP2-TB40 or left uninfected. The infection rate at day 1 p.i. was determined by flow cytometry following intranuclear staining with an IE1/2-specific antibody. Lower panels, ULBP2 surface expression on uninfected, TB40 (black lines) or ULBP2-TB40 infected cells (red line). Grey fill, isotype control. (C) Cytotoxicity assays were set up at the indicated effector/target (E/T) ratios with primary NK cells and uninfected (white circles), TB40 (black circles) and ULBP2-TB40 (red circles) infected HFF (MOI 1, 1 d.p.i.). Results are means ± SEM of 3 independent experiments performed with NK cells of one donor. (D) Compiled data of cytotoxicity assays performed with NK cells from 3 HCMV-negative donors as in (C), and in addition with ULBP2-TB40 infected target cells in the presence of an NKG2D blocking antibody (aNKG2D) or isotype antibody (iso). Results are medians (with range) of specific lysis at E/T ratio 8:1 (n = 3 donors). Data were compiled from 3 independent experiments. (E) Focus expansion assays using TB40 or ULBP2-TB40 infected HFF and primary NK cells. At day 3 of co-culturing infected cells were detected by fluorescence microscopy following staining with an IE1/2 antibody. Graph displays cumulative data as medians (with range) of experiments done with NK cells of 6 donors. A focus of infection is defined as a cluster of 3 to 60 infected cells. Four wells were analyzed per donor and all infectious foci were counted with 2 to 8 foci being present per well. Images are from one representative experiment. (F) Focus expansion assays were carried out as described in (E) and additionally in the presence of NKG2D blocking (aNKG2D) or isotype antibodies (iso) using NK cells from 4 donors. Statistical analysis for (C) (NK cell-mediated cytotoxicity assay at E/T ratio 16:1) was done with two-way ANOVA with Bonferroni correction and for (E, F) with Mann-Whitney t-test. *, P < 0.05, **, P < 0.01, ***, P < 0.001.</p

ULBP2-TB40 infected cells have lower MHC class I surface expression than TB40 infected cells.

<p>(A,B) Representative plots showing HLA class I expression using pan-HLA-A,B,C antibody (HLA I) or HLA-A02:01 antibody labeling of infected (IE1/2+) DC. Graphs provide (A) percentage and (B) median fluorescence intensity (MFI) of HLA class I positive uninfected immature DC (imDC) (white circles), or TB40 (black circles) and ULBP2-TB40 (red circles) infected DC of 4 to 6 donors. Horizontal bars represent medians. Data were analyzed with Mann-Whitney t-test. *, P < 0.05. (C) Representative staining for HLA class I on uninfected HFF or HFF infected with 1 PFU/cell of the indicated viruses at day 1 or 2 p.i. The histograms on the right depict ULBP2 expression on TB40, dUL16 TB40 (grey) or ULBP2-TB40 infected cells (red) at the indicated time points p.i. Black line, isotype controls. (D) HLA class I and ULBP2 staining gated on ULBP2-TB40 infected fibroblasts (IE1/2-positive cells) derived from two different donors.</p